Anti-proliferation domain of human Bcl-2 and DNA encoding the same

ABSTRACT

A domain of Bcl-2 that suppresses apoptosis by allowing cell survival permits cell proliferation when mutated. The wild type domain includes amino acid residues 51 to 97 (SEQ ID NO:13) of Bcl-2. Peptides including the domain and nucleotides encoding the domain are useful in molecular screening of human tumors for the presence of mutations that allow proliferation of cells that were otherwise marked for apoptosis. The peptides are also useful to screen for proteins that play a role in the modulation of cellular proliferation.

[0001] This invention was made with government support under grantsCA-33616 and CA-31719 from the National Cancer Institute. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of cellphysiology, and more particularly to tumorigenesis and to apoptosis,i.e. programmed cell death. The novel peptides and nucleotides of theinvention are useful in molecular screening of human tumors for thepresence of mutations that allow the proliferation of cells that wereotherwise marked for apoptosis. The novel peptides and nucleotides arealso useful to screen for proteins that play a role in the modulation ofcellular proliferation.

BACKGROUND OF THE INVENTION

[0003] The bcl-2 gene was discovered as typically involved in thet(14;18) chromosomal translocations observed in human follicularlymphoma (1-3). This chromosomal rearrangement results in deregulatedhigh-level expression of the bcl-2 gene. In addition, Bcl-2 is alsoexpressed at elevated levels in a variety of other tumors (4-6). TheBcl-2 protein suppresses apoptosis induced by a multitude of stimuli(7,8). Suppression of apoptosis by Bcl-2, while allowing cell survival,is characterized by growth arrest associated with Bcl-2 activity (40).Although bcl-2 was discovered as a candidate oncogene, conventionaltransformation assays indicate that it does not possess dominantoncogenic activity (9). It is therefore believed that unlike otheroncogenes, bcl-2 contributes to oncogenesis primarily by extending cellviability, thereby perturbing the homeostatic mechanisms that controlcell number and by providing an environment for other genetic changes(10).

[0004] In spite of a lack of detectable autonomous transformingactivity, bcl-2 has been shown to synergize with c-myc in the generationof malignant cells (11). Since constitutive expression of c-myc inducesapoptosis under certain conditions (12-14) that can be suppressed byBcl-2 (14-16), it appears that the c-myc-cooperating oncogenic activityof bcl-2 may be related to its anti-apoptosis activity. In addition,Bcl-2 can also efficiently suppress apoptosis induced by tumorsuppressor proteins such as p53 (17-21). This suggests that Bcl-2 maycontribute to oncogenesis by suppressing apoptosis induced by oncogenesand tumor suppressor genes.

[0005] Although mutations within the Bcl-2 protein that permitproliferation of cells that would otherwise undergo total apoptosiscould play a more direct role (as opposed to deregulated expression) inoncogenesis, thus far no such mutants have been identified in naturallyarising tumors or under experimental conditions.

SUMMARY OF THE INVENTION

[0006] The present inventor here describes the identification andcharacterization of a hitherto unrecognized domain within human Bcl-2,which the inventor has designated the “anti-proliferation (AP) domain”,that is required for the proliferation-restraining activity of Bcl-2.Mutants in this domain of Bcl-2 are described that retain the ability tosuppress apoptosis induced by the p53 tumor suppressor protein and Myconco-protein, while allowing concomitant cell proliferation.

[0007] More specifically, the present inventor has identified a deletionmutant of Bcl-2 that has a novel activity. The deletion mutant,designated Bcl2Δ51-85, not only suppresses apoptosis induced by thetumor suppressor protein p53 and the Myc onco-protein, but unlike wtBcl-2, permits continued cell proliferation. These results may haveimportant implications for oncogenesis involving Bcl-2. Unlike otheroncogenes, the bcl-2 proto-oncogene promotes cell survival withoutsignificant cell proliferation.

[0008] These results suggest that certain mutations can inactivate aproliferation-restraining activity. Further, the observed effect againstoncogene/anti-oncogene-induced apoptosis may potentially prove to be ofconsiderable significance in oncogenic events involving Bcl-2. Suchinactivating mutations within the non-conserved region of Bcl-2 mayenhance tumorigenesis by antagonizing the apoptotic activities of p53and Myc as well as by permitting continued cell proliferation.

[0009] The molecular basis for the loss of proliferation-restrainingactivity in the Bcl-2 mutant has been partially elucidated as describedin Example 3. The results suggest that the loss of activity does notcorrelate with the ability of Bcl-2 to interact with several proteins.However, the interaction between the Bcl-2 mutant and thedeath-promoting protein Bax appears to be enhanced compared to theinteraction of Bax with wild type Bcl-2. It is not clear whether thisenhancement is due to an increased affinity of the Bcl-2 mutant for Baxor increased stability of the Bcl-2/Bax complex. The importance of theBcl-2/Bax interaction to the proliferation-restraining function of Bcl-2is unknown.

[0010] Also, the region deleted in Bcl2Δ51-85 contains several Ser andThr residues. It has been reported that Bcl-2 activity can be modulatedby phosphorylation (34, 46, 47, and 49). Analysis of the activity ofseveral Bcl-2 mutants containing amino acid substitutions at Ser or Thrresidues, as described in Example 4, suggests that modulation of theproliferation-restraining activity by phosphorylation is possible.Alternative explanations to account for the mutant phenotype are alsopossible. The deleted region is rich in Ala and Pro residues.Substitution of Pro residues in two positions within the AP domainresulted in Bcl-2 mutants that permit enhanced cell proliferation. Thepossibility that these residues play some negative regulatory role inBcl-2 activity remains to be investigated.

[0011] In one aspect then, the invention provides isolatedoligonucleotides that encode the Bcl-2 AP domain or fragments of thedomain. The oligonucleotides and short segments thereof are useful forscreening for mutations in the Bcl-2 AP domain by methods known in theart, such as single strand conformational polymorphism (SSCP) and PCRmismatch analysis.

[0012] In another aspect, the present invention is directed toidentifying protein/protein interactions between the Bcl-2 AP domain andknown or as yet unidentified cellular proteins. The Bcl-2 AP domain isalso useful in the identification and cloning of genes whose proteinproducts interact with this domain in Bcl-2. The interacting proteinsmay play a role in modulation of cellular proliferation.

[0013] The present invention also relates to an isolated polypeptidethat is the Bcl-2 AP domain and fragments of the domain. The domain maybe a target for allosteric regulators of Bcl-2 function, such as proteinkinases and/or phosphatases. Accordingly, peptides derived from thisdomain, prepared synthetically or as bacterially expressed fusionproteins, can be used as substrates to identify and characterizepotential regulatory kinases and/or phosphatases.

[0014] The invention further provides screening methods to identifymolecules that modulate the proliferation-restraining activity of the APdomain. In one aspect, such screening methods involve the effect of aputative modulating molecule on the short term or long termproliferation of cells in culture expressing the AP domain. In anotheraspect, putative modulating molecules can be identified by screening foragents that disrupt necessary protein/protein interactions mediated bythe AP domain, using in vitro binding assays.

[0015] In yet another aspect, the invention provides for expressionvectors containing genetic sequences, hosts transformed with suchexpression vectors, and methods for producing the AP domain andfragments of the domain that hinder or completely block proliferation.

[0016] In additional aspects, the present invention relates toantibodies that specifically bind to the AP domain and fragments of thedomain that hinder or completely block cell proliferation. Peptidescomprising the domain are useful for producing antibodies thereto. Suchantibodies are useful for detecting and isolating proteins comprisingthe AP domain in biological specimens including, for example, cells fromall human tissues including heart tissue, lung tissue, tumor cells,brain tissue, placenta, liver, skeletal muscle, kidney, and pancreas, aswell as for modulating the proliferation-restraining activity ofproteins comprising the AP domain, in and from such biologicalspecimens, and constitute additional aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the domain structure of Bcl-2. The various conserveddomains (BH1-4) are indicated. BH1-3 are conserved among bothsurvival-promoting and death-promoting members of the Bcl-2 family ofproteins. BH1 and 2 are described in ref. 33. BH3 is described in ref.32. BH4 is conserved among survival-promoting members and corresponds tobox A described by Reed and coworkers (ref. 38). TM indicatestransmembrane domain. NH-1 indicates the E1B nineteen K homology domain(21). The amino acid sequence (SEQ ID NO:1) deleted in mutant Bcl2Δ51085is indicated.

[0018]FIGS. 2A to 2E illustrate suppression of p53-induced apoptosis byBcl-2. FIG. 2A shows an immunoprecipitation analysis of Bcl-2 andBcl2Δ51-85 expression in BRK-p53val135-E1A cells. FIG. 2B is a graphshowing survival/proliferation of BRK-p53val135-E1A cells at 32.5° C. ,pRcCMV vector; ▪, wt Bcl-2; ▴, Bcl2Δ51-85. FIGS. 2C-E show the growth ofcolonies of cells transfected with vectors carrying various Bcl-2 genes.The Figures illustrate the long-term proliferation of BRK-p53val135-E1Acells. FIG. 2C, pRcCmV vector; FIG. 2D, wt Bcl-2; FIG. 2E, Bcl2Δ51-85.

[0019]FIGS. 3A and 3B show suppression of Myc-induced apoptosis byBcl-2. FIG. 3A shows the results of an immunoprecipitation analysis ofBcl-2 and Bcl2Δ51-85 expression in Rat1MycER-Hygro cells. FIG. 3B is agraph showing survival/proliferation of RatMycER-Hygro cells. , pRcCMVvector; ▪, wt Bcl-2; ▴, Bcl2Δ51-85.

[0020]FIGS. 4A and B show the interaction of Bax with Bcl-2 andBcl2Δ51-85. BSC40 cells were transfected with pTM-HA Bax and pTM-Bcl-2or pTM-Bcl2Δ51-85 and infected with vaccinia virus vTF7-3. ³⁵S-labeledproteins were immunoprecipitated either with HA mouse monoclonalantibody (FIG. 4A) or Bcl-2 rabbit polyclonal antibody (FIG. 4B) andanalyzed on 13% SDS-polyacrylamide gels. FIG. 5 is a graph showingsurvival of BRK-p53val153-EIA cells expressing point mutants of Bcl-2.The number of viable cells was determined at various times aftershifting to 32.5° C. by trypan blue exclusion and is plotted as apercentage of the number of live cells at the start of the experiment.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Isolated, in the context of the invention, indicates that someintervention occurs that increases the level of purity of a moleculeover that found in nature.

[0022] As mentioned above, the present invention is based upon thediscovery of a heretofore unidentified domain of the human Bcl-2protein. This “anti-proliferation” or “AP” domain is required formodulation of cell proliferation. More specifically, the AP domaincompletely blocks cell proliferation.

[0023] The nucleotide sequence of human Bcl-2 according to thisinvention is based on those described in reference 41, Genbank Accession#X06487 and in reference 42, Genbank Accession #M13994. The Bcl-2nucleotide sequence described in reference 41 has a G at position 189and an A at position 287. The Bcl-2 nucleotide sequence described inreference 42 contains an alternate nucleotide (C in place of G) atposition 189 and an alternate nucleotide (G in place of A) at position287 resulting in an amino acid change at residue 96 of Thr to Ala. Thenucleotide sequence for Bcl-2 reported by Cleary et al (51), GenbankAccession #M14745, contains an alternate nucleotide at position 175 (Ain place of C), resulting in an amino acid change at residue 59 of Proto Thr.

[0024] As used herein, the phrase “anti-proliferation (or AP) domain”means a truncated human Bcl-2 protein comprising amino acid residue 51to any of amino acid residues 85-97. (SEQ ID NOS:1-13).

[0025] Thus, in addition to the core residues, i.e. residues 51 to anyof amino acid residues 85-97, the AP domain can include stretches of 1or more amino acids in the amino-terminal direction from residue 85and/or 1 or more amino acids in the carboxyl-terminal direction fromresidue 97, provided that the protein is truncated. That is, the APdomain of the present invention is not intended to include thefull-length Bcl-2 protein. For example, the AP domain can include thecore residues, i.e., residue 51 to any of amino acid residues 85-97,and/or 5, 10, 15, 20 residues, and so on, in increments of 5 to theamino-terminus of residue 51 and/or carboxyl-terminus of residue 97.

[0026] The sequences of the polypeptide that make up the core residues,i.e. SEQ ID NOS:1-13, are set forth in the section following theexamples. The sequences of any additional stretches upstream ordownstream of the core residues may be ascertained from the literature(E.G. 41, 42, 51) and protein databases such as EMBL.

[0027] Further, the data in Example 5 herein indicates that certainamino acids are needed to maintain full or partial anti-proliferationactivity of the AP domain. For example, any one of Ser at position 51,Pro at position 57, Ser at position 62, Thr-Ser at positions 69 and 70,Thr at position 74, and Pro at position 75 may contribute to theanti-proliferation function of the AP domain which is lost when residues51-85 are deleted. Thus, fragments of the AP domain that include any oneor more of the residues that fully or partially restoresanti-proliferation activity are within the present invention.

[0028] Functional equivalents of the polypeptide that make up the coreresidues as defined by SEQ ID NOS: 1-13 are also within the presentinvention. By “functional equivalent” is meant a peptide possessing abiological activity or immunological characteristic substantiallysimilar to that of the polypeptides that make up the core residues, andis intended to include “variants”, “analogs”, “homologs”, or “chemicalderivatives” possessing such activity or characteristics. Functionalequivalents of the polypeptides that make up the core residues, then,may not share an identical amino acid sequence, and conservative ornon-conservative amino acid substitutions of conventional orunconventional amino acids are possible. However, in the presentinvention any of Ser at position 51, Pro at position 57, Ser at position62, Thr and Ser at positions 69 and 70, Thr at position 74, or Pro atposition 75, may not be Ala.

[0029] Reference herein to “conservative” amino acid substitution isintended to mean the interchangeability of amino acid residues havingsimilar side chains. For example, glycine, alanine, valine, leucine andisoleucine make up a group of amino acids having aliphatic side chains;serine and threonine are amino acids having aliphatic-hydroxyl sidechains; asparagine and glutamine are amino acids having amide-containingside chains; phenylalanine, tyrosine and tryptophan are amino acidshaving aromatic side chains; lysine, arginine and histidine are aminoacids having basic side chains; and cysteine and methionine are aminoacids having sulfur-containing side chains. Interchanging one amino acidfrom a given group with another amino acid from that same group would beconsidered a conservative substitution. Preferred conservativesubstitution groups include asparagine-glutamine, alanine-valine,lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine.

[0030] Functional equivalents that possess immunological characteristicssubstantially similar to that of the polypeptides that make up the coreresidues are useful, for example, as an antigen for raising antibodiesagainst the AP domain or fragments thereof or for detection orpurification of antibodies against the AP domain or fragments thereof.

[0031] The nucleotide sequences that encode the AP domain as definedherein are also within the present invention. The nucleic acidcompositions of the invention will generally be in RNA or DNA forms,mixed polymeric forms, or any synthetic nucleotide structure capable ofbinding in a base-specific manner to a complementary strand of nucleicacid. The described nucleic acid embodiment is typically derived fromgenomic DNA or cDNA, prepared by synthesis, or derived from combinationsthereof, including polymerase chain reaction (PCR) products.

[0032] The oligonucleotides that encode the core amino acids are thosebounded by nucleotide 151 to any of nucleotides 255-291 (SEQ ID NO:14-50), where nucleotide 1 is the first nucleotide of the codon encodingthe first amino acid of Bcl-2. In these sequences the nucleotide atposition 175 can be C or A, the nucleotide at position 189 can be C orG, and the nucleotide at position 287 can be A or G.

[0033] The oligonucleotide sequences that encode the core residues, i.e.SEQ ID NOS: 14-50, are set forth in the section following the examples.The cDNA sequences toward the 5′ end of nucleotide 151 and toward the 3′end of nucleotide 291 may be ascertained from the literature (E.G. 22,42, 51) as well as from sequence databases such as Genbank.

[0034] Oligonucleotide fragments of oligonucleotide sequences thatencode the AP domain are also included within the present invention andinclude fragments that contain at least one codon encoding an amino acidneeded to maintain full or partial anti-proliferation activity of the APdomain. Examples are fragments that retain any one of the codons definedby nucleotides 151-153 (coding for Ser 51), 169-171 (coding for Pro 57),nucleotides 184-186 (coding for Ser 62), 204-210 (coding for Thr 69 andSer 70), 220-222 (coding for Thr 74), and 223-225 (coding for Pro 75).

[0035] The instant oligonucleotides and polypeptides may be obtained asdescribed herein, such as by recombinant means. For example, nucleotidesequences encoding the AP domain polypeptides or fragments thereof ofthe invention may be inserted into a suitable DNA vector, such as aplasmid, and the vector used to transform a suitable host. Therecombinant AP polypeptide or fragment is produced in the host byexpression. The transformed host may be a prokaryotic or eukaryoticcell, including a mammalian cell. The instant oligonucleotides andpolypeptides may also be used to obtain homologous nucleic acids andproteins by hybridization, for example, an instant nucleic acid can beused as a probe of a gene bank to identify clones with suitable homologytherewith. Also, within the confines of available technology, theoligonucleotides may be synthesized in vitro using, for example, solidphase oligonucleotide and oligopeptide synthetic methods known in theart.

[0036] The present invention also includes fusion polypeptides betweenthe AP domain, or fragments thereof, or truncated wt Bcl-2 polypeptidesincluding the AP domain, and other proteins or polypeptides. Forexample, fusions may include proteins that serve as purificationtargets, such as, but not limited to glutathione S-transferase (GST)(43) and the FLAG epitope tag (Eastman Kodak). In addition, fusions mayinclude polypeptides that may have amino acid residues that have been orcan be chemically modified by phosphorylation, biotinylation, acylation,or other moieties, using methods known in the art. Fusion polypeptideswill typically be made by using either synthetic polypeptide orrecombinant nucleic acid methods known in the art.

[0037] The functional importance of the AP domain is related to itsability to regulate cell proliferation. This regulation may be mediatedby one or more protein/protein interactions between the domain and knownBcl-2 interacting proteins such as Bax (44), Nip1-3 (29), Bik (32), Bak(31), R-ras (52), BAG-1 (45) and c-raf-1 (30) (see also Example 4) or asyet unidentified cellular proteins. The polypeptides of the presentinvention are useful to screen for proteins that interact with the APdomain, and these proteins and cDNA's encoding these proteins are alsopart of the invention. Such molecules are useful as agents formodulation of tumorigenesis and apoptotic activity of cells.

[0038] Methods for screening for proteins that interact with the APdomain are well known in the art and include the yeast two-hybrid system(39, 28) and expression cloning strategies using recombinant fusionproteins. (53, 54)

[0039] The in vivo genetic strategy designated ‘two hybrid’ cloning (39,28) permits rapid genetic screening in yeast of molecules thatassociate, and the method has been used to isolate from expressionlibraries cDNA clones that code for proteins interacting with severalknown proteins.

[0040] Briefly, the method relies on the double transformation of yeasthosts with plasmids that encode fusion proteins. One plasmid carriespartial sequences for a reporter molecule, for example, the GAL4 DNAbinding domain, at the amino terminus of the fusion protein andsequences for the known protein, to which a ligand is sought, also knownas the “bait” at the carboxyl-terminus. For example, the bait can be theAP domain polypeptide.

[0041] The second plasmid comprises sequences encoding a complementaryprotein for the reporter molecule, in the above case, required by theGAL4 DNA binding domain, such as the GAL4 activation domain, at theamino terminus and expressed products of individual cDNA from a bank atthe carboxyl-terminus. A suitable host is used to enable the selectionplanned. In the scenario discussed, the host would be one wherein theexpression of β-galactosidase is under the control of the GALL promoter.

[0042] Selection of double transformants are those that expressβ-galactosidase, hence would be blue colonies on an X-gal plate becausethe bait protein encoded by the cDNA of the second plasmid bind and thatinteraction juxtaposes the two GAL4 regulatory elements required forβ-galactosidase expression.

[0043] An additional related strategy is to isolate positive clones fromthe two hybrid assay that interact with GAL4 DNA-binding domain-Bcl-2(wt) fusion but not with a GAL4 DNA-binding domain-Bcl-2Δ51-85 fusion.Such interacting proteins may require the identified domain for theirinteraction.

[0044] Thus, the present invention provides a method for screening for apolypeptide that binds the AP domain of Bcl-2 protein, the methodcomprising:

[0045] (a) conducting a double transformation wherein one vectorexpresses a fusion protein comprising the AP domain or a fragmentthereof and a reporter molecule and the other vector expresses a fusionprotein comprising a complementary protein for the reporter molecule andthe polypeptide to be screened;

[0046] (b) monitoring for activation of the reporter molecule; and

[0047] (c) isolating cDNA that encodes the protein that binds to the APdomain or the fragment thereof,

[0048] wherein the AP domain or fragment thereof is a truncated Bcl-2protein comprising residues 51 to any of residues 85-97 (SEQ IDNOS:1-13) or a fragment thereof that contains at least one amino acidneeded to maintain full or partial anti-proliferation activity of the APdomain.

[0049] In a related embodiment, the present invention also provides amethod of screening for a polypeptide that binds the AP domain of Bcl-2protein, the method comprising:

[0050] (a) conducting a first double transformation wherein one vectorexpresses a fusion protein comprising the AP domain and a reportermolecule and the other vector expresses a fusion protein comprising acomplementary protein for the reporter molecule and the polypeptide tobe screened;

[0051] (b) conducting a second double transformation wherein one vectorexpresses a fusion protein comprising Bcl-2 with the AP domain or afragment of Bcl-2 that contains at least one amino acid needed tomaintain full or partial anti-proliferation activity of the AP domaindeleted and a reporter molecule and the other vector expresses a fusionprotein comprising a complementary protein for the reporter molecule andthe polypeptide to be screened;

[0052] (c) monitoring for activation of the reporter molecule in bothdouble transformations; and

[0053] (d) isolating cDNA that encodes a polypeptide that binds in step(a) but not in step (b).

[0054] In a second example of methods of screening for proteins thatinteract with the AP domain, a cDNA encoding Bcl-2 residues 51-85 iscloned into an E.coli expression vector that will encode a glutathioneS-transferase (GST)-Bcl-2 domain fusion protein.

[0055] The fusion protein is isolated following expression in bacteriaand radiolabeled for use as a probe to screen for cDNA of proteinscapable of interacting with the AP domain from a human cell λ-phageexpression library. (53, 54) Briefly, a λ-phage expression library (e.g.λ-ZAP, Stratagene) is plated on E.coli and resulting plaques aretransferred to isopropyl-β-D-thogalactoside (IPTG)-impregnatednitrocellulose filters to induce protein expression. ³²P-radiolabeledGST-AP domain fusion proteins or unlabelled GST-AP domain fusionproteins that can be detected with an anti-GST antibody, are used as aprobe to screen for expressed proteins capable of interacting with theAP domain. Positive clones can be isolated and the gene encoding aprotein capable of interacting with the AP domain can be sequenced andcharacterized.

[0056] Thus, the present invention provides a method for screening for apolypeptide that interacts with the AP domain of Bcl-2 protein, themethod comprising:

[0057] (a) expressing cDNA that encodes a polypeptide to be screened;

[0058] (b) immobilizing the expressed polypeptide; and

[0059] (c) detecting interaction with a polypeptide comprising the APdomain or fragment thereof;

[0060] wherein the AP domain or fragment thereof is a truncated Bcl-2protein comprising residues 51 to any of residues 85-97 (SEQ IDNOS:1-13) or a fragment thereof that contains at least one amino acidneeded to maintain full or partial anti-proliferation activity of the APdomain.

[0061] Alternatively, the biochemical isolation of interacting moleculesis also possible using isolated polypeptides comprising the Bcl-2 APdomain. For example, GST-AP domain fusion proteins can be immobilized onglutathione (GSH)-agarose columns to capture interacting proteins fromcell lysates. Cell lysates from BRK-p53val135-E1A cells are passed overthe column. Following washing to remove non-binding proteins,interacting proteins can be eluted using GSH or other conditions knownto disrupt protein/protein interactions such as salt, pH, guanidine HCl,or detergent gradients. Eluted proteins can be identified, for example,by SDS-PAGE and microsequencing. If necessary, oligonucleotide probesbased on the protein sequence can be used to clone the correspondinggene from an appropriate cDNA library.

[0062] Thus, the present invention provides a method for screening for apolypeptide that interacts with the AP domain of Bcl-2 protein, themethod comprising:

[0063] (a) immobilizing a polypeptide comprising the AP domain orfragment of the AP domain;

[0064] (b) contacting the immobilized polypeptide with putativeinteracting protein; and

[0065] (c) identifying interacting protein;

[0066] wherein the AP domain or fragment thereof is a truncated Bcl-2protein comprising residues 51 to any of residues 85-97 (SEQ IDNOS:1-13) or a fragment thereof that contains at least one amino acidneeded to maintain full or partial anti-proliferation activity of the APdomain.

[0067] The present invention includes the use of the AP domain orfragments for the identification of agents that modulate AP domainmediated functions. Such agents may include peptides comprising the APdomain or mutants of the AP domain or comprising an AP domain. A“mutant” as used herein refers to a peptide having an amino acidsequence that differs from the amino acid sequence of the naturallyoccurring peptide or protein by at least one amino acid. Mutants mayhave the same biological and immununological activity as the naturallyoccurring AP domain. However, the biological or immunological activityof mutants may differ or be lacking. Identification of such agents canbe accomplished by the screening of peptide or compound libraries, orother information banks, in assays for agonists or antagonists thatenhance or inhibit AP domain function, e.g. survival-promoting andproliferation-restraining activity, as well as protein binding.

[0068] For example, BRK-p53val135-E 1 A cells expressing Bcl-2 or atruncated version of Bcl-2 comprising the AP domain can be used toscreen for agents that inhibit the proliferation-restraining activitythe AP domain detected by increased proliferation in the short termassay and/or allowing colony formation in the long term assay.

[0069] In another example, agents can be identified that modulate theproliferation-restraining activity of the AP domain by screening forcompounds that influence protein/protein interactions mediated by the APdomain using an in vitro binding assay. In such as an assay, a GSTfusion protein comprising the AP domain is immobilized to GSH-agarose.Binding of a radiolabaled-interacting protein in the presence of one ormore compounds to be tested would be quantitated by scintillationcounting. Inhibitors of the interaction would result in a decrease inassociated interacting protein. For rapid-throughput screening, theGST/AP-domain fusion protein and biotinylated interacting protein areused in a multi-well plate format. Biotinylated proteins can beexpressed and isolated from E.coli using PinPoint vectors (Promega) byknown methods. The purified biotinylataed protein is immobilized on aneutravidin-coated plate and binding of the GST/Ap-domain fusion proteinin the presence of test compounds is detected by ELISA using an anti-GSTmonoclonal antibody. Inhibitors of the interaction would score as adecreased ELISA signal.

[0070] A high speed screen using immobilized or “tagged” combinatoriallibraries can be used to identify agents that bind directly to the APdomain. Such agents are candidates to be tested for their ability toenhance or inhibit the proliferation-restraining activity of Bcl-2.

[0071] The AP domain may be a target for allosteric regulators of Bcl-2function such as protein kinases and/or phosphatases. Phosphorylation ofBcl-2 has been reported (46-49) and it has been suggested thatphosphorylation/dephosphorylation may play a role in the regulation ofBcl-2 function. The identified domain of Bcl-2 contains severalpotential phosphorylation sites. Thus, the polypeptides of the presentinvention comprising the AP domain can be used as substrates to measurean enzymatic activity, such as kinase or phosphatase. In this aspect, invitro kinase assays are carried out by incubating cell lysates, such asderived from BRK-p53val135-E1A cells, with AP domain polypeptides,prepared synthetically or as bacterially expressed fusion proteins, inthe presence of ²³P-labeled ATP in 10 mM Tris buffer containing 10 mMMgCl₂ and 1 μM unlabeled ATP. Phosphatase activity is detected byincubating cell lysates with phosphorylated AP domain polypeptide,derived from in vitro kinase assays described above or isolated fromcells, and following the release of radiolabeled phosphate from the APdomain. Purification and sequencing of the protein responsible for thisactivity can be accomplished by standard methods such as those describedin “Protein Purification: Principles and Practice,” by Robert Scopes(Ed: C. Cantor, Springer Verlag, Heidelberg, 1982).

[0072] Synthetic peptides or fusion proteins containing this domain canbe used for immunizing animals in the production of polyclonal ormonoclonal antibodies that bind to this domain in Bcl-2. Such antibodieswould be useful as reagents for studying the function of this domain.For example, microinjection of anti-domain antibodies may alter the cellcycle arrest activity of Bcl-2. Such antibodies may also prove to beuseful in screening for mutations in this domain of Bcl-2 that causealterations in antibody binding. These mutations may correlate withalterations in Bcl-2 function.

[0073] The AP polypeptides of the invention also may be used for thedetection of Bcl-2 by means of standard assays includingradioimmunoassays and enzyme immunoassays.

[0074] The polypeptides of the present invention or fusion proteinsthereof are also useful to make antibodies for detection ordetermination of proteins comprising the AP domain, for example, infractions from tissue/organ excisions, by means of immunochemical orother techniques in view of the antigenic properties thereof.

[0075] Immunization of animals with polypeptides comprising the APdomain alone or in conjunction with adjuvants by known methods canproduce antibodies specific for the AP domain polypeptide. Antiserumobtained by conventional procedures may be utilized for this purpose.For example, a mammal, such as a rabbit, may be immunized with a peptidecomprising the AP domain, thereby inducing the formation of polyclonalantibodies thereagainst. Monoclonal antibodies also may be generatedusing known procedures.

[0076] If the target molecule is poorly immunogenic, known methods forenhancing immunogenicity, such as, use of adjuvants, use of fragments ofthe target molecule as antigen, conjugating the target molecule orfragments thereof to a known carrier, such as albumin or keyhole limpethemocyanin, immunizing immune cells in vitro and the like, as known inthe art can be used.

[0077] Antibodies against the AP domain polypeptides or fragmentsthereof of the invention may be used to screen cDNA expression librariesfor identifying clones containing cDNA inserts encoding structurallyrelated, immunocrossreactive proteins that may be members of an APdomain family of proteins. Screening of cDNA and mRNA expressionlibraries is known in the art. Similarly, antibodies against AP domainpolypeptides or fragments thereof can be used to identify or purifyimmunocrossreactive proteins related to this domain, or to detect ordetermine the amount of proteins containing the AP domain in a cell orcell population, for example, in tissue or cells, such as lymphocytes,obtained from a patient. Known methods for such measurements includeimmunopreciptiation of cell extracts followed by PAGE, in situ detectionby immunohistochemical methods, and ELISA methods, all of which are wellknow in the art. In addition, antibodies against the AP domain orfragments thereof may be used to modulate the proliferation-restrainingactivity of proteins comprising the AP domain.

[0078] Accordingly, the present invention also provides an isolatedantibody that binds to the AP domain of Bcl-2 and a hybridoma that makesmonoclonal antibody that specifically binds to the AP domain.

[0079] The cDNA of the present invention may be used for screening formutations in the AP domain in, for example, human tumors. Indeed,mutations within this domain associated with non-Hodgkin's lymphomashave been reported including a change in the nucleotide (T in place ofC) at position 175 resulting in a substitution of Pro 59 with Ser (50).

[0080] Methods for screening for such mutations have been described, andinclude single strand conformational polymorphism (SSCP) of polymerasechain reaction-amplified DNA fragments (SSPC-PCR) (55, 56) andPCR-mismatch analysis (50, 51).

[0081] In SSCP-PCR, oligonucleotide primers are used to amplify thesegment of the Bcl-2 gene encoding the AP domain from DNA or mRNAisolated from a test sample or from cDNA made from the test sample. ThePCR product is then heat denatured, subjected to electrophoresis onpolyacrylamide gels and transferred to a nylon membrane. The fragmentcan be detected by a chemiluminescence detection system and the relativemobility of the test fragment with a control fragment from wt Bcl-2 isdetermined. A single base change can be detected by this method.

[0082] Accordingly, the present invention provides a method of screeningfor mutations in the AP domain of Bcl-2, the method comprising:

[0083] (a) isolating genomic DNA, cDNA or mRNA from a specimen to bescreened;

[0084] (b) amplifying DNA fragments encoding the AP domain or portionsthereof from the genomic DNA, cDNA or mRNA;

[0085] (c) denaturing the amplified product;

[0086] (d) subjecting the denatured product to electrophoresis; and

[0087] (e) detecting mutations by comparing the mobility of thedenatured amplified product to a control DNA encoding the AP domain orportions thereof corresponding positionally to the DNA fragmentsamplified in step (b);

[0088] wherein the control DNA encoding the AP domain or portionsthereof is from the truncated cDNA encoding the bcl-2 gene and fragmentsof the truncated cDNA.

[0089] Alternatively, in PCR-mismatch analysis, PCR products from thetest sample are mixed with radiolabeled PCR products from the wild typeBcl-2 AP domain. The mixed PCR material is denatured and then annealed.Chemical modification and cleavage of heteroduplexes containingmismatched nucleotides is analyzed by gel electrophoresis. PCR-generatedDNAs containing mutations are then subcloned and sequenced to identifythe precise nature of the mutation.

[0090] Thus, the present invention provides a method of screening formutations in the AP domain of Bcl-2, said method comprising:

[0091] (a) isolating genomic DNA, cDNA or mRNA from a specimen to bescreened;

[0092] (b) amplifying DNA fragments encoding the AP domain or portionsthereof from the genomic DNA, cDNA or mRNA;

[0093] (c) mixing the amplified product with labeled PCR product fromthe corresponding position in a control AP domain or portion thereof;

[0094] (d) denaturing and annealing the mixed PCR products; and

[0095] (e) analyzing for mismatched nucleotides by electrophoresisfollowing chemical modification;

[0096] wherein the control DNA encoding the AP domain or portionsthereof is selected from the truncated cDNA encoding the bcl-2 gene andfragments of the truncated cDNA.

[0097] In a related embodiment, the present invention also provides amethod of screening for mutations in the AP domain of Bcl-2, said methodcomprising:

[0098] (a) isolating genomic DNA, cDNA or mRNA from a specimen to bescreened;

[0099] (b) amplifying DNA fragments encoding the AP domain or portionsthereof from the genomic DNA, cDNA or mRNA; and

[0100] (c) sequencing the amplified DNA product.

[0101] Of course, the polynucleotide sequences of the invention may beused in the PCR method to detect the presence of mRNA encoding AP domainpolypeptides in for, example, cells from all human tissues includingheart tissue, lung tissue, tumor cells, brain tissue, placenta, liver,skeletal muscle, kidney, and pancreas.

EXAMPLES

[0102] The invention will now be described by means of working examplesthat are not intended to be limiting.

[0103] Materials and Methods

[0104] Plasmids. Plasmid pRcCMV-Bcl-2 was constructed by cloning thehuman bcl-2 gene (22) into the HindIII and XbaI sites of the mammalianexpression vector pRcCMV (Invitrogen). Mutant Bcl2Δ51-85 was constructedby PCR mutagenesis using a mutagenic oligonucleotide primer5′-GGA-CCA-CAG-GTG-GCA-CCG-GGC-TGA-GGC-TAG-CGG-AGA-AGA-AGC-CCG-GTG-CGG-GGG-CG-3′(SEQ ID NO:51) and two other primers complementary to the 5′ and 3′ endsof bcl-2. This mutagenesis introduces an NheI site, and substitutes analanine and a serine residue in the deleted region. The PCR product wascloned into the HindIII and XbaI sites of pRcCMV to generatepRcCMV-Bcl2Δ51-85. pTM1-based plasmids expressing wt Bcl-2 and mutantBcl2Δ51-85 were constructed by cloning the respective genes into theNcoI and SalI sites of the vector pTM1 (23).

[0105] Cell lines. The BRK-p53val135-E1A cell line has been described(21) and was maintained at 38.5° C. in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% fetal calf serum. BRK-p53val135-E1A cellsstably expressing Bcl-2 were generated by transfection of variouspRcCMV-based Bcl-2 expression plasmids and selection with G418 (250μg/ml)(GIBCO/BRL). Rat1a and Rat1MycER-Hygro cells have been described(14,24). Cells expressing ER fusion proteins were maintained in DMEMmedia without phenol red and 10% fetal calf serum (certified lowestrogen content, GIBCO/BRL). Rat1MycER-Hygro cells expressing Bcl-2were selected by transfection with pRcCMV-Bcl2 or pRcCMV-Bcl2Δ51-85 andselection with 400 μg/ml G418. DNA transfections were carried out by thestandard calcium phosphate method.

[0106] Cell death assays. BRK-p53val135-E1A cells were plated at 5×10⁵cells/35 mm dish. After 12 hours at 38.5° C., the dishes were shifted to32.5° C., and at various intervals cells were trypsinized intriplicates, stained with 0.2% trypan blue and viable cells werecounted. Similarly 5×10⁵ Rat1-Hygro cells were plated in 35 mm dishes,incubated for 12 hours at 37° C., washed three times in serum-free DMEMand maintained in fresh media containing 0.1% fetal calf serum and 1 μMβ-estradiol. Viable cell number was determined at various intervals.

[0107] Immunoprecipitation. Bcl-2 or Bcl2Δ51-85 proteins wereco-expressed with HA epitope-tagged Bax using the vaccinia virus/T7coupled expression system as previously described (29). BSC40 cells weretransfected with pTM1 expression plasmids using LipofectAMINE(GIBCO/BRL) and infected with the recombinant vaccinia virus vTF7-3 (23)expressing the T7 RNA polymerase. Sixteen hours post-infection, cellswere metabolically labeled with 500 μCi of ³⁵S-methionine and -cysteinemixture for two hours and lysed in isotonic buffer (29) containingprotease inhibitors (0.04 mg/ml aprotinin, 0.2 mg/ml leupeptin, 200 μMphenylmethylsulfonyl fluoride). Lysates were precleared with proteinA-Sepharose for 1 hour, which was removed by centrifugation. Theproteins were immunoprecipitated with a rabbit polyclonal antibodyspecific for human Bcl-2 or with HA monoclonal antibody (12CA5;Boehringer Mannheim). The proteins were analyzed by electrophoresis on13% SDS polyacrylamide gels and detected by fluorography.

EXAMPLE 1 Effect on p53-Induced Apoptosis

[0108] A non-conserved region located between residues 51 and 85 wasexamined (FIG. 1) with the rationale that such sequences may regulatethe activity of Bcl-2.

[0109] Deletion of this region of Bcl-2 (Bcl2Δ51-85) did notsignificantly alter the level of expression of the mutant protein (FIGS.2A; 3A). The effect of Bcl-2 wt and mutant Bcl2Δ51-85 on apoptosisinduced by the tumor suppressor protein p53 (25) was tested. Baby ratkidney (BRK) cells transformed with adenovirus E1A and a ts mutant ofp53 (p53val135) (26) express very high levels of mutant p53 at thenon-permissive (38.5° C.) temperature and undergo rapid apoptosis afterthe p53 protein assumes wt conformation at 32.5° C. (27). This apoptosiscan be efficiently suppressed by Bcl-2 (20). BRK-p53val135-E1A cellswere transfected with pRcCMV vector or pRcCMV-Bcl-2 or pRcCMV-Bcl2Δ51-85and G418 resistant colonies were selected at 38.5° C. As expected, wtBcl-2 efficiently suppressed cell death compared to cells transfectedwith pRcCMV vector (FIG. 2A). Cells expressing Bcl2Δ51-85 did not losecell viability significantly at 32.5° C. Surprisingly, however, thesecells also proliferated efficiently at this temperature in contrast tocells expressing wt Bcl-2 (FIG. 2B). Deletion of additional residues,form 51-98, resulted in a mutant, Bcl-2Δ51-98 that was unable tosuppress cell death in this assay (FIG. 5, Table 1) suggesting thatresidues between 85 and 98 may be critical for Bcl-2 survival function.

[0110] The effect of mutant Bcl2Δ51-85 on long term proliferation wasalso determined. Pooled cell lines transfected with Bcl-2 wt orBcl2Δ51-85 or pRcCMV vector were plated at low cell density, maintainedat 32.5° C. for three weeks and stained with Giemsa (FIGS. 2C-2E). Cellstranfected with pRcCMV died rapidly without forming any detectablecolonies. Cells tranfected with wt Bcl-2 survived for an extendedperiod, but formed very few proliferating colonies. Consistent withtheir behavior in short term cell survival/proliferation assays (FIG.2B), cells transfected with mutant Bcl2Δ51-85 formed numerousproliferating colonies. These results indicate that the mutantBcl2Δ51-85 facilitates long term proliferation of cells under conditionsthat otherwise result in apoptosis.

EXAMPLE 2 Effect on Myc-Induced Apoptosis

[0111] The effect of Bcl2Δ51-85 on Myc-induced apoptosis was alsotested. Rat1 cells expressing the c-myc gene fused to the human estrogenreceptor (c-mycER-Hygro) undergo apoptosis after Myc expression isactivated by addition of β-estradiol and cells are deprived of serum(13,14). The c-mycER-Hygro cells were tranfected with pRcCMV vector orpRcCMV-based plasmids expressing wt Bcl-2 or mutant Bcl2Δ51-85 andpooled G418-resistant cell lines were established. Immunoprecipitation(FIG. 3A) and protein-blot (not shown) analyses revealed that thevarious Rat1 cell lines expressed comparable levels of wt or the mutantBcl-2 proteins. The effect of Bcl-2 expression on Myc-induced apoptosiswas then determined by treating the cells with 1 μM f-estradiol in mediacontaining 0.1% fetal calf serum.

[0112] Deregulated Myc expression induced significant cell death.Expression of wt Bcl-2 resulted in about 60% cell survival. As in thecase of BRK/p53val135-E1 A cells, the expression of Bcl2Δ51-85 mutantnot only suppressed cell death but also induced significantproliferation on mycER-Hygro cells in low serum after a lag period ofabout one day.

EXAMPLE 3 Interaction of Cellular Proteins with Bcl2Δ51-85

[0113] In order to determine if deletion of the amino acid regionencompassing residues 51-85 affected interaction of various cellularproteins, the interaction of several cellular proteins that have beenpreviously reported to interact with Bcl1-2 either by two-hybridinteraction studies in yeast (28) or by co-immunoprecipitation analyseswas examined. In these studies, no major difference was observed in thepatterns of interaction of Nip1-3 (29), c-Raf-1 (30), R-ras (52), Bak(31), and Bik (32) (not shown). In contrast, the level of interactionbetween Bax (33) and Bcl2Δ51-85 appeared to be significantly enhanced incomparison to wt bcl-2 in co-immunoprecipitation assays (FIGS. 4A and4B). This enhanced interaction appears to be significant consideringthat the total level of Bax was similar in cells expressing eitherBcl2Δ51-85 or wt Bcl-2.

EXAMPLE 4 Characterization of Critical Residues Within the Bcl-2 Residue51-85 Domain

[0114] In an effort to characterize critical residues within the Bcl-2residue 51-85 domain, several Bcl-2 mutants encoding single amino acidsubstitutions were constructed and tested for their effect on cellsurvival and proliferation. In the short term survival assay (FIG. 5 andTable 1), none of the point mutants gave an enhanced proliferationactivity comparable to the Bcl-2D51-85 mutant, though two mutants, P75Aand S51 A, had some effect. While most of the point mutations resultedin Bcl-2 molecules that retained at least significant survival function,substitution of serine at position 62 with alanine completely abolishedsurvival activity. This result demonstrates that this region hassubstantial influence on the survival function of Bcl-2 as well asmodulation of proliferation. In the long term assay (Table 1), severalof the point mutants permitted significant colony formation, suggestingthat these residues may contribute to the proliferation-restrainingactivity of Bcl-2. One substitution mutant, S51A, had a hyperprotectiveeffect that was apparent in the long term assay. With wild type Bcl-2,the BRK-p53val135-E1 A cells eventually die when subjected to theprolonged exposure at 32.5° C. used in the long term assay. In contrast,cells expressing Bcl-2 S51A survived for the duration of the assay,though no significant colony formation was observed. TABLE 1 Comparisonof survival activity and long term colony formation for Bcl-2 mutants.Survival activity and long-term proliferation (colony formation) wasmeasured in BRK-p53val135-E1a cells at 32.5° C. as described for FIG. 2.Δ indicates deleted residues, substitution mutations (such as S51A) areindicated by the amino acid changed followed by the position number andthe substituted amino acid. For survival activity, + is normal +\− ispartial, ++ and +++ are above normal. For colony formation, + is small,++ is medium, +++ is large colonies. Mat indicates that cells werepresent without obvious colony formation. −, indicates no cellsremaining. Survival colony Bcl-2 mutant Activity formation vector − −wild type + − Δ51-85 ++ +++ Δ51-98 − − S51A +++ mat T56A +\− − P57A+\− + S62A − − TS69-70AA +\− + T74A +\− ++ P75A ++ +

[0115] A straightforward interpretation of these results is that theeffect of the Bcl-2Δ51-85 mutant is a sum of the hyperproliferativefunction of the S51A mutant and the proliferative effects of the P57A,TS69-70AA, T74A, and P75A mutants.

Sequences

[0116] Polypeptide and nucleotide sequences referred to herein by SEQ IDNOS. are listed below.

[0117] In the polypeptide sequences (Pro/Thr) and (Thr/Ala) means thatthe amino acid at that position can be Pro or Thr and Thr or Ala,respectively.

[0118] In the nucleic acid sequences M represents A or C, S represents Cor G, and R represents A or G. Ser Gln Pro Gly His Thr Pro His (Pro/Thr)Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:1) Pro Leu GlnThr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala. Ser Gln Pro Gly His ThrPro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ IDNO:2) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu.Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro ValAla Arg Thr Ser (SEQ ID NO:3) Pro Leu Gln Thr Pro Ala Ala Pro Gly AlaAla Ala Gly Pro Ala Leu Ser. Ser Gln Pro Gly His Thr Pro His (Pro/Thr)Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:4) Pro Leu GlnThr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro. Ser Gln ProGly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg ThrSer (SEQ ID NO:5) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala GlyPro Ala Leu Ser Pro Val. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) AlaAla Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:6) Pro Leu Gln ThrPro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro. Ser GlnPro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala ArgThr Ser (SEQ ID NO:7) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala AlaGly Pro Ala Leu Ser Pro Val Pro Pro. Ser Gln Pro Gly His Thr Pro His(Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:8) ProLeu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro ValPro Pro Val. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser ArgAsp Pro Val Ala Arg Thr Ser (SEQ ID NO:9) Pro Leu Gln Thr Pro Ala AlaPro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val. Ser GlnPro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala ArgThr Ser (SEQ ID NO:10) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala AlaGly Pro Ala Leu Ser Pro Val Pro Pro Val Val His. Ser Gln Pro Gly His ThrPro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ IDNO:11) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala LeuSer Pro Val Pro Pro Val Val His Leu. Ser Gln Pro Gly His Thr Pro His(Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:12) ProLeu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro ValPro Pro Val Val His Leu (Thr/Ala). Ser Gln Pro Gly His Thr Pro His(Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:13) ProLeu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro ValPro Pro Val Val His Leu (Thr/Ala) Leu. TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC (SEQ ID NO:14) CAGGACCTCG CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC (SEQ ID NO:15) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCG TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC(SEQ ID NO:16) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ IDNO:17) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:18)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:19)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCATCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:20)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:21)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:22)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:23)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:24)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:25)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:26)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:27)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTG TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ IDNO:28) CAGGACCTCG CCGCTGGAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ IDNO:29) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGCC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ IDNO:30) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGCCA TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQID NO:31) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA C TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC (SEQ ID NO:32) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CC TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC (SEQ ID NO:33) CAGGACCTCG CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCT TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:34) CAGGACCTCG CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTG TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:35) CAGGACCTCGCCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:36)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGCCA CCTGTG TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC(SEQ ID NO:37) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA CCTGTGGT TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC (SEQ ID NO:38) CAGGACCTCG CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTC TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:39) CAGGACCTCGCCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCACCTGTGGTCC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQID NO:40) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA CCTGTGGTCC A TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC (SEQ ID NO:41) CAGGACCTCG CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC AC TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:42) CAGGACCTCGCCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCACCTGTGGTCC ACC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC(SEQ ID NO:43) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCT TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC (SEQ ID NO:44) CAGGACCTCG CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTG TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:45) CAGGACCTCGCCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCACCTGTGGTCC ACCTGR TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC(SEQ ID NO:46) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTGRC TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:47) CAGGACCTCG CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCCACCTGRCC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ IDNO:48) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGCCA CCTGTGGTCC ACCTGRCCC TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC (SEQ ID NO:49) CAGGACCTCG CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTGRCCCTTCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:50)CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGCCA CCTGTGGTCC ACCTGRCCCT C

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[0176]

1 51 35 amino acids amino acid <Unknown> linear protein 1 Ser Gln ProGly His Thr Pro His Xaa Ala Ala Ser Arg Asp Pro Val 1 5 10 15 Ala ArgThr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 25 30 Gly ProAla 35 36 amino acids amino acid <Unknown> linear protein 2 Ser Gln ProGly His Thr Pro His Xaa Ala Ala Ser Arg Asp Pro Val 1 5 10 15 Ala ArgThr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 25 30 Gly ProAla Leu 35 37 amino acids amino acid <Unknown> linear protein 3 Ser GlnPro Gly His Thr Pro His Xaa Ala Ala Ser Arg Asp Pro Val 1 5 10 15 AlaArg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 25 30 GlyPro Ala Leu Ser 35 38 amino acids amino acid <Unknown> linear protein 4Ser Gln Pro Gly His Thr Pro His Xaa Ala Ala Ser Arg Asp Pro Val 1 5 1015 Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 2530 Gly Pro Ala Leu Ser Pro 35 39 amino acids amino acid <Unknown> linearprotein 5 Ser Gln Pro Gly His Thr Pro His Xaa Ala Ala Ser Arg Asp ProVal 1 5 10 15 Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly AlaAla Ala 20 25 30 Gly Pro Ala Leu Ser Pro Val 35 40 amino acids aminoacid <Unknown> linear protein 6 Ser Gln Pro Gly His Thr Pro His Xaa AlaAla Ser Arg Asp Pro Val 1 5 10 15 Ala Arg Thr Ser Pro Leu Gln Thr ProAla Ala Pro Gly Ala Ala Ala 20 25 30 Gly Pro Ala Leu Ser Pro Val Pro 3540 41 amino acids amino acid <Unknown> linear protein 7 Ser Gln Pro GlyHis Thr Pro His Xaa Ala Ala Ser Arg Asp Pro Val 1 5 10 15 Ala Arg ThrSer Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 25 30 Gly Pro AlaLeu Ser Pro Val Pro Pro 35 40 42 amino acids amino acid <Unknown> linearprotein 8 Ser Gln Pro Gly His Thr Pro His Xaa Ala Ala Ser Arg Asp ProVal 1 5 10 15 Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly AlaAla Ala 20 25 30 Gly Pro Ala Leu Ser Pro Val Pro Pro Val 35 40 43 aminoacids amino acid <Unknown> linear protein 9 Ser Gln Pro Gly His Thr ProHis Xaa Ala Ala Ser Arg Asp Pro Val 1 5 10 15 Ala Arg Thr Ser Pro LeuGln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 25 30 Gly Pro Ala Leu Ser ProVal Pro Pro Val Val 35 40 44 amino acids amino acid <Unknown> linearprotein 10 Ser Gln Pro Gly His Thr Pro His Xaa Ala Ala Ser Arg Asp ProVal 1 5 10 15 Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly AlaAla Ala 20 25 30 Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His 35 4045 amino acids amino acid <Unknown> linear protein 11 Ser Gln Pro GlyHis Thr Pro His Xaa Ala Ala Ser Arg Asp Pro Val 1 5 10 15 Ala Arg ThrSer Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala 20 25 30 Gly Pro AlaLeu Ser Pro Val Pro Pro Val Val His Leu 35 40 45 46 amino acids aminoacid <Unknown> linear protein 12 Ser Gln Pro Gly His Thr Pro His Xaa AlaAla Ser Arg Asp Pro Val 1 5 10 15 Ala Arg Thr Ser Pro Leu Gln Thr ProAla Ala Pro Gly Ala Ala Ala 20 25 30 Gly Pro Ala Leu Ser Pro Val Pro ProVal Val His Leu Xaa 35 40 45 47 amino acids amino acid <Unknown> linearprotein 13 Ser Gln Pro Gly His Thr Pro His Xaa Ala Ala Ser Arg Asp ProVal 1 5 10 15 Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly AlaAla Ala 20 25 30 Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu XaaLeu 35 40 45 104 base pairs nucleic acid single linear DNA 14 TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGC 104 105 base pairs nucleic acidsingle linear DNA 15 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCG 105 106 base pairs nucleic acid single linear DNA 16 TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGC 106 107 base pairs nucleic acidsingle linear DNA 17 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCT 107 108 base pairs nucleic acid single linear DNA 18 TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTC 108 109 base pairs nucleicacid single linear DNA 19 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCA 109 110 base pairs nucleic acid single linear DNA 20TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG 110 111 basepairs nucleic acid single linear DNA 21 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG C 111 112 base pairs nucleic acid single linearDNA 22 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CC 112 113base pairs nucleic acid single linear DNA 23 TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCC 113 114 base pairs nucleic acidsingle linear DNA 24 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCG 114 115 base pairs nucleic acid single linear DNA 25TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGG 115 116base pairs nucleic acid single linear DNA 26 TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGT 116 117 base pairs nucleic acidsingle linear DNA 27 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTG 117 118 base pairs nucleic acid single linear DNA 28TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGC 118 119base pairs nucleic acid single linear DNA 29 TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCC 119 120 base pairs nucleicacid single linear DNA 30 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA 120 121 base pairs nucleic acid single linear DNA31 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 C121 122 base pairs nucleic acid single linear DNA 32 TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CC 122 123base pairs nucleic acid single linear DNA 33 TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCT 123 124 base pairsnucleic acid single linear DNA 34 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTG 124 125 base pairs nucleicacid single linear DNA 35 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSGACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGCCTGCGCTCAG CCCGGTGCCA 120 CCTGT 125 126 base pairs nucleic acid singlelinear DNA 36 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGCCAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAGCCCGGTGCCA 120 CCTGTG 126 128 base pairs nucleic acid single linear DNA37 TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120CCTGTGGT 128 129 base pairs nucleic acid single linear DNA 38 TCCCAGCCCGGGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGACCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTC 129130 base pairs nucleic acid single linear DNA 39 TCCCAGCCCG GGCACACGCCCCATMCAGCC GCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGCCCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC 130 131 basepairs nucleic acid single linear DNA 40 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC A 131 132 base pairsnucleic acid single linear DNA 41 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC AC 132 133 base pairsnucleic acid single linear DNA 42 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACC 133 134 base pairsnucleic acid single linear DNA 43 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCT 134 135 base pairsnucleic acid single linear DNA 44 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTG 135 136 base pairsnucleic acid single linear DNA 45 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTGR 136 137 basepairs nucleic acid single linear DNA 46 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTGRC 137 138 basepairs nucleic acid single linear DNA 47 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTGRCC 138 139 basepairs nucleic acid single linear DNA 48 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTGRCCC 139 140 basepairs nucleic acid single linear DNA 49 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTGRCCCT 140 141 basepairs nucleic acid single linear DNA 50 TCCCAGCCCG GGCACACGCC CCATMCAGCCGCATCCCGSG ACCCGGTCGC CAGGACCTCG 60 CCGCTGCAGA CCCCGGCTGC CCCCGGCGCCGCCGCGGGGC CTGCGCTCAG CCCGGTGCCA 120 CCTGTGGTCC ACCTGRCCCT C 141 56 basepairs nucleic acid single linear other nucleic acid /desc =“Oligonucleotide Primer” 51 GGACCACAGG TGGCACCGGG CTGAGGCTAG CGGAGAAGAAGCCCGGTGCG GGGGCG 56

What is claimed:
 1. A truncated bcl-2 gene comprising nucleotides 151 toany one of nucleotides 255-291 (SEQ ID NOS:14-50), and fragments of thetruncated bcl-2 gene that contain at least one codon encoding an aminoacid needed to maintain full or partial anti-proliferation activity ofthe AP domain.
 2. The truncated bcl-2 gene of claim 1, wherein thefragments contain at least one of nucleotides 151-153, nucleotides169-171, nucleotides 184-186, nucleotides 204-210, nucleotides 220-222,and nucleotides 223-225.
 3. The truncated bcl-2 gene of claim 1, whichcomprises nucleotides 151 to any one of nucleotides 255-291 (SEQ ID NO:14-50).
 4. The truncated bcl-2 gene of claim 1, which comprisesnucleotides 151-255. (SEQ ID NO:14).
 5. The truncated bcl-2 gene ofclaim 1, which consists of nucleotides 151 to any one of nucleotides255-291 (SEQ ID NO:14-50).
 6. The truncated bcl-2 gene of claim 1, whichconsists of nucleotides 151-255 (SEQ ID NO:14).
 7. RNA complementary tothe truncated bcl-2 gene of anyone of claims 1-6.
 8. A truncated Bcl-2protein comprising residues 51 to any of 85-97 (SEQ ID NOS: 1-13) andfragments of the truncated Bcl-2 protein that contain at least one aminoacid needed to maintain full or partial anti-proliferation activity ofthe AP domain.
 9. The truncated Bcl-2 protein of claim 8, wherein thefragments contain at least one of Ser residue 51, Pro residue 57, Serresidue 62, Thr and Ser residues 69 and 70, Thr residue 74, and Proresidue
 75. 10. The truncated Bcl-2 protein of claim 8 comprisingresidues 51-85 (SEQ ID NO:1) and fragments of the truncated proteincomprising residues 51-85 that contain at least one of Ser residue 51,Pro residue 57, Ser residue 62,.Thr and Ser residues 69 and 70, Thrresidue 74, and Pro residue
 75. 11. The truncated Bcl-2 protein of claim8 which consists of residues 51 to any of 85-97 (SEQ ID NOS:1-13). 12.The truncated Bcl-2 protein of claim 8 which consists of residues 51-85(SEQ ID NO:1).
 13. A method of screening for mutations in the AP domainof Bcl-2, said method comprising: (a) isolating genomic DNA, cDNA ormRNA from a specimen to be screened; (b) amplifying DNA fragmentsencoding the AP domain or portions thereof from the genomic DNA, cDNA ormRNA (c) denaturing the amplified product; (d) subjecting the denaturedproduct to electrophoresis; and (e) detecting mutations by comparing themobility of the denatured amplified product to a control DNA encodingthe AP domain or portions thereof corresponding positionally to the DNAfragments amplified in step (b); wherein said control DNA encoding theAP domain or portions thereof is selected from the truncated bcl-2 geneand fragments thereof claimed in claim
 1. 14. The method of claim 13,wherein truncated bcl-2 gene extends from nucleotide no. 151—nucleotideno. 255 (SEQ ID NO:14).
 15. A method of screening for mutations in theAP domain of Bcl-2, said method comprising: (a) isolating genomic DNA,cDNA or mRNA from a specimen to be screened; (b) amplifying DNAfragments encoding the AP domain or portions thereof from the genomicDNA, cDNA or mRNA; (c) mixing the amplified product with labeled PCRproduct from the corresponding position in a control AP domain orportion thereof; (d) denaturing and annealing the mixed PCR products;and (e) analyzing for mismatched nucleotides by electrophoresisfollowing chemical modification; wherein the control DNA encoding the APdomain or portion thereof is selected from the truncated bcl-2 gene andfragments thereof claimed in claim
 1. 16. The method of claim 15,wherein truncated bcl-2 gene extends from nucleotide no. 151—nucleotideno. 255 (SEQ ID NO:14).
 17. A method of screening for mutations in theAP domain of Bcl-2, said method comprising: (a) isolating genomic DNA,cDNA or mRNA from a specimen to be screened; (b) amplifying DNAfragments encoding the AP domain or portions thereof from the genomicDNA, cDNA or mRNA; and (c) sequencing the amplified DNA product.
 18. Anisolated cDNA comprising a sequence that encodes a polypeptide thatbinds in a double transformation to a truncated Bcl-2 protein defined byresidues 51 to any of residues 85-97 (SEQ ID NOS:1-13) and fragmentsthereof that contain at least one amino acid needed to maintain full orpartial anti-proliferation activity of the AP domain.
 19. Thepolypeptide encoded by the isolated cDNA of claim
 18. 20. A method forscreening for a polypeptide that binds the AP domain of Bcl-2 protein,said method comprising: (a) conducting a double transformation whereinone vector expresses a fusion protein comprising the AP domain or afragment thereof and a reporter molecule and the other vector expressesa fusion protein comprising a complementary protein for the reportermolecule and said polypeptide to be screened; (b) monitoring foractivation of the reporter molecule; and (c) isolating cDNA that encodesthe protein that binds to said AP domain or said fragment thereof,wherein said AP domain or fragment thereof is a truncated Bcl-2 proteincomprising residues 51 to any of 85-97 (SEQ ID NOS:1-13) or a fragmentthereof that contains at least one amino acid needed to maintain full orpartial anti-proliferation activity of the AP domain.
 21. An isolatedcDNA comprising a sequence that encodes a polypeptide that binds wtbcl-2 in a double transformation and does not bind Bcl-2Δ51 to 85-97 ordeletions of a fragment of a bcl-2 gene that contains at least one codonencoding an amino acid needed to maintain full or partialanti-proliferation activity of the AP domain.
 22. A polypeptide encodedby the isolated cDNA of claim
 21. 23. A method of screening for apolypeptide that binds the AP domain of Bcl-2 protein, said methodcomprising: (a) conducting a first double transformation wherein onevector expresses a fusion protein comprising the AP domain and areporter molecule and the other vector expresses a fusion proteincomprising a complementary protein for the reporter molecule and saidpolypeptide to be screened; (b) conducting a second doubletransformation wherein one vector expresses a fusion protein comprisingBcl-2 with the AP domain or fragments of Bcl-2 that contain at least oneamino acid needed to maintain full or partial anti-proliferationactivity of the AP domain deleted and a reporter molecule and the othervector expresses a fusion protein comprising a complementary protein forthe reporter molecule and said polypeptide to be screened; (c)monitoring for activation of the reporter molecule in both doubletransformations; and (d) isolating cDNA that encodes a polypeptide thatbinds in step (a) but not in step (b).
 24. A method for screening for apolypeptide that interacts with the AP domain of Bcl-2 protein, themethod comprising: (a) expressing cDNA that encodes a polypeptide to bescreened; (b) immobilizing the expressed polypeptide; and (c) detectinginteraction with a polypeptide comprising the AP domain or fragmentthereof wherein the AP domain or fragment thereof is a truncated Bcl-2protein comprising residues 51 to any of residues 85-97 (SEQ IDNOS:1-13) or a fragment thereof that contains at least one amino acidneeded to maintain full or partial anti-proliferation activity of the APdomain.
 25. A method for screening for a polypeptide that interacts withthe AP domain of Bcl-2 protein, the method comprising: (a) immobilizinga polypeptide comprising the AP domain or fragment of the AP domain; (b)contacting the immobilized polypeptide with putative interactingprotein; and (c) identifying interacting protein; wherein the AP domainor fragment thereof is a truncated Bcl-2 protein comprising residues 51to any of residues 85-97 (SEQ ID NOS:1-13) or a fragment thereof thatcontains at least one amino acid needed to maintain full or partialanti-proliferation activity of the AP domain.
 26. An isolated antibodythat binds to the AP domain of Bcl-2 or fragments of Bcl-2 that containat least one amino acid needed to maintain full or partialanti-proliferation activity of the AP domain.
 27. The isolated antibodyof claim 27, wherein the AP domain consists of Bcl-2 residue 51 to anyof residues 85-97 (SEQ ID NO:1-13) or fragments of the AP domain thatcontain at least one of Ser residue 51, Pro residue 57, Ser residue 62,Thr and Ser residues 69 and 70, Thr residue 74, and Pro residue
 75. 28.The isolated antibody of claim 26, wherein the AP domain consists ofBcl-2 residues 51-85 (SEQ ID NO:1).
 29. The isolated antibody of any oneof claims 26, 27 or 28, wherein the antibody is a monoclonal antibodythat specifically binds to the AP domain.
 30. A hybridoma that makes themonoclonal antibody of claim
 29. 31. A method of producing isolated APdomain or fragments thereof, said method comprising: (a) constructing avector comprising DNA encoding the AP domain or fragments thereofcontaining at least one codon encoding an amino acid needed to maintainfull or partial anti-proliferation activity of the AP domain; (b)transforming a suitable host cell with said vector of step (a); (c)culturing said host cell under conditions that allow expression of saiddomain or fragments thereof by said host cell; and (d) isolating saiddomain or fragment thereof expressed by said host cell of step (c). 32.The method of claim 31, wherein said host cell is a mammalian cell.